Detection of integrated insertion nucleotide sequences is important in many contexts. For example, insertion nucleotide sequences can transport oncogenes in mammals, antibiotic resistance genes in bacteria and genes conveying identifiable traits in plants. Bacterial antibiotic resistance is a major worldwide clinical problem and public health concern (see, for example, Sheldon, Clin Lab Sci (2005) 18:170 and French, Adv Drug Deliv Rev (2005) 57:1514). Clearly, an efficient, sensitive and reliable method for detecting the presence or absence of an integrated insertion nucleotide is valuable in clinical diagnostics and other contexts.
Others have developed methods for detecting integrated insertion nucleotide sequences. In one approach, the integrated insertion nucleotide is detected using PCR where one primer hybridizes to the target nucleic acid sequence and the other primer hybridizes to the insertion nucleotide. Positive detection of the amplicon indicates the presence of an insertion polynucleotide. This method can result in false negatives, or undetected insertion nucleotides, because the sequences of insertion nucleotides are often variable. Available primers may or may not hybridize to the insertion polynucleotide. See, for example, the IDI-MRSA™ diagnostic assay Test by GeneOhm Sciences, San Diego, Calif.
In another approach, the integrated insertion nucleotide is detected using PCR where both primers hybridize to the insertion polynucleotide. Again, positive detection of amplicon indicates the presence of an integrated insertion polynucleotide. This method also has the shortcoming that it can result in false negatives, due to the polymorphic nature of the integrated insertion nucleotides. Also, it is not clear whether the insertion polynucleotide is integrated into the target nucleic acid sequence when both forward and reverse primers hybridize to the insertion polynucleotide. See, for example, Kreiswirth, et al. J Clin Microbiol (2005) 43:4585 and LIGHTCYCLER, thermocycler, MRSA Detection Kit by Roche Diagnostics, Alameda, Calif.
In a further approach, presence or absence of the integrated insertion polynucleotide is identified using PCR where one primer hybridizes to the target nucleic acid sequence and the other primer hybridizes to a sequence straddling the integration site between the target nucleic acid sequence and the insertion polynucleotide. Here, negative detection (lack of amplification) of amplicon indicates the presence of an integrated insertion polynucleotide. This method has the disadvantage that a negative signal indicates the positive integration of an insertion polynucleotide (see, U.S. Pat. No. 6,156,507).
There remains a need for efficient, sensitive methods for detecting integrated insertion polynucleotides which provide a positive signal indicative of the integration of the insertion polynucleotide.